Process for preparing L-amino acids

ABSTRACT

A process for preparing L-amino acids employing coryneform bacteria in which the AmtR regulator has been attenuated is provided. Recombinant bacteria, polynucleotides and vectors corresponding to or having the attenuated AmtR regulator are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 12/468,512 filed May19, 2009, which claims priority to U.S. Provisional Application No.61/147,271, filed Jan. 26, 2009, and German Application No.102008001874.0 filed May 20, 2008, the disclosures of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a process for preparing L-amino acidscomprising a recombinant coryneform bacteria in which the AmtR regulatorhas been attenuated.

2. Discussion of the Background

L-Amino acids are used in human medicine, in the pharmaceuticalindustry, in the food industry and very particularly in livestocknutrition.

It is known that L-amino acids such as, for example, L-lysine areprepared by fermentation of strains of coryneform bacteria, especiallyCorynebacterium glutamicum. Because of their great importance, work toimprove the methods used to prepare L-amino acids has been continuous.Process improvements may relate to fermentation technology measures suchas, for example, stirring and supplying oxygen, or to the composition ofthe nutrient media, such as, for example, the sugar concentration duringfermentation, or to the working up to product form by, for example ionexchange chromatography, or to the intrinsic output properties of themicroorganism itself.

The methods used for improving the output properties of thesemicroorganisms include mutagenesis, followed by selection and choice ofmutants. The strains obtained in this way are resistant toantimetabolites or are auxotrophic for metabolites of regulatoryimportance, and produce L-amino acids. A known antimetabolite is thelysine analogue S-(2-aminoethyl)-L-cysteine (AEC).

Methods of recombinant DNA technology have likewise been used for strainimprovement of L-amino acid-producing strains of the genusCorynebacterium, especially Corynebacterium glutamicum. These methodshave been directed to modifying individual amino acid biosynthesis genesand investigating the effect on amino acid production.

Reference sources describing the biology, genetics and biotechnology ofCorynebacterium glutamicum include in the “Handbook of Corynebacteriumglutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis,2005), the special edition of the Journal of Biotechnology (ChiefEditor: A. Pühler) entitled “A New Era in Corynebacterium glutamicumBiotechnology” (Journal of Biotechnology 104/1-3, (2003)) and the bookby T. Scheper (Managing Editor) “Microbial Production of L-Amino Acids”(Advances in Biochemical Engineering/Biotechnology 79, Springer Verlag,Berlin, Germany, 2003).

The nucleotide sequence of the genome of Corynebacterium glutamicumATCC13032 is described by Ikeda and Nakagawa (Applied Microbiology andBiotechnology 62, 99-109 (2003)), in EP 1 108 790 and by Kalinowski etal. (Journal of Biotechnology 104(1-3), (2003)). The nucleotide sequenceof the genome of Corynebacterium glutamicum R is described by Yukawa etal. (Microbiology 153(4):1042-1058 (2007)).

The nucleotide sequence of the genome of Corynebacterium efficiens isdescribed by Nishio et al. (Genome Research. 13 (7), 1572-1579 (2003)).

The nucleotide sequences of the genome of Corynebacterium glutamicum andCorynebacterium efficiens are likewise available in the database of theNational Center for Biotechnology Information (NCBI) of the NationalLibrary of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan(DDBJ, Mishima, Japan) or in the nucleotide sequence database of theEuropean Molecular Biologies Laboratories (EMBL, Heidelberg, Germany orCambridge, UK).

The structural definition of the Corynebacterium glutamicum genome madeit possible inter alia to carry out wide-ranging investigations on themetabolism and regulatory network of this bacterium (Silberbach andBurkovski, Journal of Biotechnology 126(1): 101-110 (2006)).

An essential precondition for synthesizing amino acids and in generalfor growing the cells is an appropriate supply of nitrogen. C.glutamicum is able to utilize various nitrogen sources, includingammonium, L-glutamic acid, glutamine and urea. Depending on theconcentration and nature of the available nitrogen source, particularenzymes and transport systems are synthesized and activated. For energyreasons, strict regulation is necessary (“nitrogen control”). Theregulation of gene expression and the global signal transduction in thenitrogen metabolism of C. glutamicum has been investigated in detail byvarious authors.

The expression of nitrogen-regulated genes is regulated in C. glutamicumby the global repressor AmtR. When the nitrogen supply is good, AmtRrepresses expression of the genes of the amt-soxA-ocd operon, of thegltBD operon, of the amtB-glnK-glnD operon and of glnA and crnT genes(Jacoby et al., Molecular Microbiology 37: 964-977 (2000); Beckers etal., Microbiology, 147: 2961-2170 (2001); Nolden et al., FEMSMicrobiological Letters 201: 91-98 (2001)). It was possible in furtherinvestigations (Beckers et al., Journal of Bacteriology 186(22): 7645-52(2004); Beckers et al., Molecular Microbiology 58(2): 580-595 (2005))inter alia to show an AmtR-dependent regulation also for the genes ofthe gluABCD operon, of the NCgl1915-1918 operon, of the urtABCDE operon,of the ureABCEFGD operon, and the codA gene and the NCgl1099 gene.

EP 1 460 128 reports on the effect of deleting the amtR gene in a ΔargRstrain on the production of various amino acids.

OBJECT OF THE INVENTION

Even after all the work described, a need to improve methods of L-aminoacid production, in terms of efficiency, yield and purity remains. Thisand other objects have been achieved by the present invention, the firstembodiment of which includes: a recombinant, L-amino acid-secreting,coryneform bacterium comprising an amtR gene which codes for an AmtRregulator wherein

-   -   an amino acid sequence of the amtR gene is at least 90%        identical to the amino acid sequence of SEQ ID NO:2 and    -   length of amino acids of the amtR gene essentially comprises 222        amino acids, and    -   the amtR gene is attenuated by at least one of the measures        selected from the group consisting of

a) replacement of the nucleobase guanine at position 7 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by thymine,

b) replacement of the nucleobase cytosine at position 11 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by guanine,

c) replacement of the nucleobase thymine at position 40 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by guanine,

d) replacement of the nucleobase thymine at position 45 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by guanine,

e) deletion of one or more of the nucleobases of position 40 to 45,preferably deletion of all nucleobases of position 40 to 45, of thepromoter region of the amtR gene shown in SEQ ID NO:5,

f) deletion of one or more of the nucleobases between position 72 and 78of the promoter region of the amtR gene shown in SEQ ID NO:5,

g) replacement of one or more of the nucleobases adenine or guaninebetween position 72 and 78 of the promoter region of the amtR gene shownin SEQ ID NO:5 by thymine or cytosine,

h) exchange of the ATG start codon at position 1 to 3 of the codingregion of the amtR gene for a GTG or TTG start codon,

i) exchange of the glycine at position 3 of the amino acid sequence ofSEQ ID NO:2 for another proteinogenic L-amino acid,

j) exchange of the L-isoleucine at position 24 of the amino acidsequence of SEQ ID NO:2 for an amino acid selected from the group ofL-asparagine, L-glutamine, L-glutamic acid and L-aspartic acid,preferably L-aspartic acid,

k) exchange of the L-leucine at position 31 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group of L-proline,L-asparagine, L-glutamine, L-phenylalanine, L-tyrosine, L-tryptophan,L-glutamic acid and L-aspartic acid, preferably L-proline,

l) exchange of the L-phenylalanine at position 32 of the amino acidsequence of SEQ ID NO:2 for an amino acid selected from the group ofglycine, L-glutamic acid, L-aspartic acid, L-proline and L-cysteine,preferably L-proline,

m) exchange of the glycine at position 36 of the amino acid sequence ofSEQ ID NO:2 for an amino acid selected from the group of L-glutamicacid, L-aspartic acid, L-isoleucine, L-histidine and L-phenylalanine,preferably L-histidine, L-glutamic acid or L-aspartic acid,

n) exchange of the L-threonine at position 42 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group of L-proline,L-isoleucine, L-methionine, L-glutamine, L-tryptophan, L-glutamic acidand L-aspartic acid, preferably L-glutamic acid,

o) exchange of the glycine at position 50 of the amino acid sequence ofSEQ ID NO:2 for an amino acid selected from the group of L-glutamicacid, L-aspartic acid, L-isoleucine, L-histidine, L-tryptophan andL-phenylalanine, preferably L-tryptophan,

p) exchange of the L-glutamine at position 53 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group of L-cysteine,L-methionine, L-tyrosine, L-tryptophan and L-phenylalanine, preferablyL-phenylalanine,

q) exchange of the L-alanine at position 54 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group ofL-phenylalanine, L-isoleucine, L-tryptophan, L-tyrosine, L-histidine,L-glutamic acid and L-aspartic acid, preferably L-histidine,

r) exchange of the L-serine at position 55 of the amino acid sequence ofSEQ ID NO:2 for an amino acid selected from the group of L-proline,L-phenylalanine, L-tryptophan, L-lysine, L-arginine, L-glutamic acid andL-aspartic acid, preferably L-phenylalanine,

s) exchange of the L-tyrosine at position 57 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group of L-proline,glycine, L-methionine, L-glutamic acid and L-aspartic acid, preferablyL-aspartic acid,

t) exchange of the L-tyrosine at position 58 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group of L-proline,L-methionine and L-cysteine, preferably L-proline,

u) exchange of the L-histidine at position 59 of the amino acid sequenceof SEQ ID NO:2 for an amino acid selected from the group of L-lysine,L-aspartic acid, L-isoleucine, L-proline and glycine, preferablyL-proline, and

v) exchange of the L-lysine at position 63 of the amino acid sequence ofSEQ ID NO:2 for an amino acid selected from the group of L-alanine,L-glutamic acid, L-aspartic acid, L-asparagine, L-tyrosine andL-tryptophan, preferably L-asparagine.

a. The present invention provides a recombinant, L-amino acid-secreting,coryneform bacterium in which the amtR gene which codes for an AmtRregulator whose amino acid sequence may be at least 85% or at least 90%,preferably at least 95%, particularly preferably at least 98% or atleast 99% and very particularly preferably identical to the amino acidsequence of SEQ ID NO:2 and essentially comprises a length of 222 aminoacids has been attenuated by one or more of the measures selected fromthe group a) to v) listed above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Coryneform bacteria may be employed to produce the bacteria of theinvention. Among the coryneform bacteria, the genus Corynebacterium maybe preferred. Within the genus Corynebacterium, strains derived from thefollowing species may be preferred:

Corynebacterium efficiens, such as, for example, the type strainDSM44549,

Corynebacterium glutamicum, such as, for example, the type strainATCC13032 or the strain R, and

Corynebacterium ammoniagenes, such as, for example, the strain ATCC6871,with very particular preference for the species Corynebacteriumglutamicum.

Some representatives of the species Corynebacterium glutamicum may alsobe described by other names. These names include, for example:

Corynebacterium acetoacidophilum ATCC13870,

Corynebacterium lilium DSM20137,

Corynebacterium melassecola ATCC17965,

Brevibacterium flavum ATCC14067,

Brevibacterium lactofermentum ATCC13869, and

Brevibacterium divaricatum ATCC14020.

The term “Micrococcus glutamicus” for Corynebacterium glutamicum haslikewise been in use.

Some representatives of the species Corynebacterium efficiens have alsobeen referred to as Corynebacterium thermoaminogenes, such as, forexample, the strain from FERM BP-1539.

The strains of coryneform bacteria (starting strains) employed for theattenuation measures preferably already have the ability to enrich thedesired L-amino acid(s) in the cell or secrete them into the nutrientmedium surrounding them and accumulate them there. The term “produce” isalso used hereinafter to describe this secretion and accumulation. Inparticular, the strains of coryneform bacteria employed for theattenuation measures have the ability to enrich or accumulate in thecell or in the nutrient medium at least 0.25 g/l, preferably at least0.5 g/l, more preferably at least 1.0 g/l, most preferably at least 1.5g/l, particularly preferably at least 2.0 g/l, more particularlypreferably at least 4 g/l or most particularly preferably at least 10g/l of the desired compound in at most 120 hours, preferably at most 96hours, more preferably 48 hours, most preferably 36 hours, particularlypreferably at most 24 hours or most particularly preferably at most 12hours. The starting strains may preferably be strains which have beenproduced by mutagenesis and selection, by recombinant DNA techniques orby a combination of both methods.

It is obvious and requires no further explanation that a bacterium ofthe invention may also be acquired by firstly attenuating the amtR genein a wild strain such as, for example, in the type strain ATCC13032 orin the strain ATCC14067, with the aid of the measures of the invention,and subsequently causing the bacterium, by suitable further geneticmeasures, to produce the desired L-amino acid(s).

The term L-amino acids according to the present invention includes theproteinogenic amino acids, plus L-ornithine and L-homoserine.Proteinogenic L-amino acids include the L-amino acids which occur innatural proteins, that is to say in proteins of microorganisms, plants,animals and humans. The proteinogenic amino acids may include L-asparticacid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-arginine, L-proline, L-selenocysteine and L-pyrrolysine.The preferred L-amino acids may be L-lysine, L-glutamic acid,L-glutamine, L-arginine, L-proline and L-ornithine. L-lysine may beparticularly preferred.

The description of amino acids or L-amino acids according to the presentinvention may include the salts thereof, such as, for example, thelysine monohydrochloride or lysine sulfate in the case of the amino acidL-lysine.

Examples of known representatives of L-lysine-producing or -secretingstrains of coryneform bacteria may be:

Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in EP 0 358940,

Corynebacterium glutamicum MH20-22B (=DSM16835) described in Menkel etal. (Applied and Environmental Microbiology 55(3), 684-688 (1989)),

Corynebacterium glutamicum AHP-3 (=Ferm BP-7382) described in EP 1 108790,

Corynebacterium glutamicum NRRL B-11474 described in U.S. Pat. No.4,275,157,

Corynebacterium glutamicum DSM13994 described in U.S. Pat. No.6,783,967,

Corynebacterium glutamicum DSM16834 described in WO 06/063660,

Corynebacterium glutamicum DSM17119 described in WO 06/100211,

Corynebacterium glutamicum DSM17223 described in WO 06/125714,

Corynebacterium glutamicum DSM16937 described in WO 06/077004, and

Corynebacterium thermoaminogenes AJ12521 (=FERM BP-3304) described inU.S. Pat. No. 5,250,423.

Data on the taxonomic classification of strains of the group ofcoryneform bacteria may be found inter alia in Seiler (Journal ofGeneral Microbiology 129, 1433-1477 (1983)), Kinoshita (1985, GlutamicAcid Bacteria, p 115-142. In: Demain and Solomon (ed), Biology ofIndustrial Microorganisms. The Benjamin/Cummins Publishing Co., London,UK), Kämpfer and Kroppenstedt (Canadian Journal of Microbiology 42,989-1005 (1996)), Liebl et al (International Journal of SystematicBacteriology 41, 255-260 (1991)), Fudou et al (International Journal ofSystematic and Evolutionary Microbiology 52, 1127-1131 (2002)) and inU.S. Pat. No. 5,250,434.

Strains with the designation “ATCC” may be purchased from the AmericanType Culture Collection (Manassas, Va., USA). Strains with thedesignation “DSM” may be purchased from the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany). Strainswith the designation “NRRL” may be purchased from the AgriculturalResearch Service Patent Culture Collection (ARS, Peoria, Ill., US).Strains with the designation “FERM” may be purchased from the NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan).

L-Lysine-producing coryneform bacteria typically may have afeedback-resistant or desensitized aspartate kinase. Feedback-resistantaspartate kinases are aspartate kinases (LysC) which, by comparison withthe wild form (wild type), show less sensitivity to inhibition bymixtures of lysine and threonine or mixtures of AEC (aminoethylcysteine)and threonine or lysine alone or AEC alone. The genes or alleles codingfor these aspartate kinases which are desensitized by comparison withthe wild type may also be referred to as lysC^(FBR) alleles. NumerouslysC^(FBR) alleles coding for aspartate kinase variants which have aminoacid exchanges by comparison with the wild-type protein areconventionally known. The coding region of the wild-type lysC gene ofCorynebacterium glutamicum ATCC13032 corresponding to access numberAX756575 of the NCBI database is depicted in SEQ ID NO:7 and thepolypeptide encoded by this gene is depicted in SEQ ID NO:8. The aminoacid sequence of the wild form of aspartate kinase varies slightly indifferent wild-type strains of Corynebacterium glutamicum. Thus, theaspartate kinase of the wild-type strain Corynebacterium glutamicumATCC14067 contains alanine at position 317. The wild-type aspartatekinase of the strain ATCC 13032 contains serine at this position, asdepicted in SEQ ID NO:8.

In a preferred embodiment of the present invention, theL-lysine-producing coryneform bacteria have a lysC allele which codesfor an aspartate kinase variant which has the amino acid sequence of SEQID NO:13, and includes one or more of the amino acid exchanges selectedfrom the group consisting of:

a) LysC A279T (exchange of L-alanine at position 279 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-threonine; seeU.S. Pat. No. 5,688,671 and access numbers E06825, E06826, E08178 andI74588 to I74597),

b) LysC A279V (exchange of L-alanine at position 279 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-valine; see JP6-261766 and access number E08179),

c) LysC L297Q (exchange of L-leucine at position 297 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-glutamine; see DE102006026328,

d) LysC S301F (exchange of L-serine at position 301 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-phenylalanine; seeU.S. Pat. No. 6,844,176 and access number E08180),

e) LysC S301Y (exchange of L-serine at position 301 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-tyrosine; seeKalinowski et al. (Molecular and General Genetics 224, 317-324 (1990))and access number X57226),

f) LysC T308I (exchange of L-threonine at position 308 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-isoleucine; see JP6-261766 and access number E08181),

g) LysC T311I (exchange of L-threonine at position 311 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-isoleucine; see WO00/63388 and U.S. Pat. No. 6,893,848),

h) LysC R320G (exchange of L-arginine at position 320 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for glycine; see Jettenet al. (Applied Microbiology and Biotechnology 43, 76-82 (995)) andaccess number L27125),

i) LysC G345D (exchange of glycine at position 345 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-aspartic acid; seeJetten et al. (Applied Microbiology and Biotechnology 43, 76-82 (995))and access number L16848),

j) LysC T380I (exchange of L-threonine at position 380 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-isoleucine; see WO01/49854 and access number AX192358), and

k) LysC S381F (exchange of L-serine at position 381 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for L-phenylalanine; seeEP 0435132), with L-alanine being present where appropriate at position317 instead of L-serine.

Preferred embodiments of the present invention include the lysC^(FBR)allele lysC T311I (exchange of threonine at position 311 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for isoleucine) and alysC^(FBR) allele comprising at least one exchange selected from thegroup of A279T (exchange of alanine at position 279 of the encodedaspartate kinase protein shown in SEQ ID NO: 13 for threonine), S381F(exchange of serine at position 381 of the encoded aspartate kinaseprotein shown in SEQ ID NO: 13 for phenylalanine), with the serine atposition 317 being exchanged where appropriate for alanine (S317A).

An especially preferred embodiment of the present invention includes thelysC^(FBR) allele lysC T311I (exchange of threonine at position 311 ofthe encoded aspartate kinase protein shown in SEQ ID NO: 13 forisoleucine), with the serine at position 317 being exchanged whereappropriate for alanine (S317A).

The strain DSM 16833 (WO 06/063660) has a lysC^(FBR) allele which codesfor an aspartate kinase protein which comprises the amino acid exchangeT311I.

The strain NRRL B-11474 (U.S. Pat. No. 4,275,157) has a lysC^(FBR)allele which codes for an aspartate kinase protein which comprises theamino acid exchange S381F. It may also be advantageous for lysineproduction to overexpress the lysC^(FBR) alleles.

In a further embodiment, the L-lysine-producing bacteria of the genusCorynebacterium according to the present invention which preferablyadditionally comprise a polynucleotide which codes for alysine-insensitive aspartate kinase variant may have one or more of themeasures selected from the group consisting of:

a) overexpressed polynucleotide (dapA gene) which codes for adihydrodipicolinate synthase (DapA, EC No. 4.2.1.52),

b) overexpressed polynucleotide (asd gene) which codes for anaspartate-semialdehyde dehydrogenase (Asd, EC No. 1.2.1.11),

c) overexpressed polynucleotide (ddh gene) which codes for ameso-diaminopimelate dehydrogenase (Ddh, EC No. 1.4.1.16),

d) overexpressed polynucleotide (lysA gene) which codes for adiaminopimelate decarboxylase (LysA, EC No. 4.1.1.20),

e) overexpressed polynucleotide (aat gene) which codes for an aspartateaminotransferase (Aat, EC No. 2.6.1.1),

f) overexpressed polynucleotide (lysE gene) which codes for apolypeptide having L-lysine export activity (LysE, lysine effluxpermease),

g) overexpressed polynucleotide which codes for a pyruvate carboxylase(Pyc, EC No. 6.4.1.1), and

h) overexpressed polynucleotide (dapB gene) which codes for adihydrodipicolinate synthase (DapB, EC No. 1.3.1.26).

The bacterial genes conventionally known may be used for this purpose.The endogenous genes or polynucleotides of the genus Corynebacterium maypreferably be used, particularly preferably those of the speciesCorynebacterium glutamicum, Corynebacterium efficiens andCorynebacterium ammoniagenes and very particularly preferably those ofthe species Corynebacterium glutamicum.

Endogenous genes or polynucleotides refer to the open reading frames(ORF), genes or alleles, or polynucleotides thereof, which are presentin the population of a species.

The dapA gene of Corynebacterium glutamicum strain ATCC13032 isdescribed for example in EP 0 197 335. For overexpression of the dapAgene of Corynebacterium glutamicum it is additionally possible to employinter alia the mutations MC20 and MA16 of the dapA promoter as describedin U.S. Pat. No. 6,861,246.

The asd gene of Corynebacterium glutamicum strain ATCC 21529 isdescribed for example in U.S. Pat. No. 6,927,046.

The lysA gene of Corynebacterium glutamicum ATCC13869 (Brevibacteriumlactofermentum) is described for example in U.S. Pat. No. 6,090,597.

The ddh gene is described for example in Ishino et al. (Agricultural andBiological Chemistry 52(11), 2903-2909 (1988)).

The aat gene of Corynebacterium glutamicum ATCC13032 is described forexample in Kalinowski et al (Journal of Biotechnology 104 (1-3), 5-25(2003); see also access number NC_006958). It is referred to therein asthe aspB gene. A gene coding for an aspartate aminotransferase isreferred to as aspC in U.S. Pat. No. 6,004,773. Marienhagen et al(Journal of Bacteriology 187 (22), 7693-7646 (2005)) refer to the aatgene as aspT gene.

The lysE gene of Corynebacterium glutamicum R127 is described forexample in U.S. Pat. No. 6,858,406. It may be possible in the same wayto employ the lysE gene of the strain ATCC13032 used in U.S. Pat. No.6,861,246.

The pyc gene of Corynebacterium glutamicum of the ATCC 13032 strain isdescribed for example in WO 99/18228 and WO 00/39305. It may also bepossible to use alleles of the pyc gene described for example in U.S.Pat. No. 6,965,021. The pyruvate carboxylases described in this patenthave one or more of the amino acid exchanges selected from the group:Pyc E153D (exchange of L-glutamic acid at position 153 for L-asparticacid), Pyc A1825 (exchange of L-alanine at position 182 for L-serine),Pyc A2065 (exchange of L-alanine at position 206 for L-serine), PycH227R (exchange of L-histidine at position 227 for L-arginine), PycA455G (exchange of L-alanine at position 455 for glycine), and PycD1120E (exchange of L-aspartic acid at position 1120 for L-glutamicacid). It may likewise be possible to use the pyc allele described in EP1 108 790, which codes for a pyruvate carboxylase which comprises theamino acid exchange Pyc P458S (exchange of L-proline at position 458 forL-serine).

Overexpression conventionally refers to an increase in the intracellularconcentration or activity of a ribonucleic acid, of a protein(polypeptide) or of an enzyme by comparison with the starting strain(parent strain) or wild-type strain. A starting strain (parent strain)refers to the strain on which the measure leading to overexpression hasbeen carried out.

The increase in concentration or activity may be achieved for example byincreasing the copy number of the appropriate polynucleotideschromosomally or extra-chromosomally by at least one copy.

A conventional method for increasing the copy number consists ofincorporating the appropriate polynucleotide into a vector, preferably aplasmid, which is replicated by a coryneform bacterium. It may also bepossible to employ transposons, insertion elements (IS elements) orphages as vectors. A large number of suitable vectors are conventionallyknown.

Another widely used method for achieving overexpression may be themethod of chromosomal gene amplification. In this method, at least oneadditional copy of the polynucleotide of interest is inserted into thechromosome of a coryneform bacterium. Amplification methods of this typeare described for example in WO 03/014330 or WO 03/040373.

A further method for achieving overexpression consists of linking theappropriate gene or allele in a functional manner (operably linked) to apromoter or to an expression cassette. Suitable promoters forCorynebacterium glutamicum are described for example in FIG. 1 of thereview article by Patek et al. (Journal of Biotechnology 104(1-3),311-323 (2003)). It may be possible in the same way to employ thevariants of the dapA promoter which are described by Vasicova et al(Journal of Bacteriology 181, 6188-6191 (1999)), for example thepromoter A25. A further possibility may be to use the gap promoter ofCorynebacterium glutamicum (EP 06007373). Finally, the T3, T7, SP6, M13,lac, tac and trc promoters are described by Amann et al. (Gene 69(2),301-315 (1988)) and Amann and Brosius (Gene 40(2-3), 183-190 (1985)). Apromoter of this type may be inserted for example upstream of therelevant gene, typically at a distance of approximately 1-500nucleobases from the start codon.

In alternative embodiments of the present invention, the overexpressionmeasures increase the activity or concentration of the appropriatepolypeptide by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%or 500%, up to a maximum of 1000% or 2000%, based on the activity orconcentration of the polypeptide in the strain before the measureleading to the overexpression.

It may be likewise possible, in addition to the measures relating to theamtR gene, to attenuate or switch off individual biosynthesis genes.

In alternative embodiments of the present invention, improving theproduction of L-lysine, L-valine or L-isoleucine, preferably L-lysine,may be achieved by attenuation or switching off one or more of the genesselected from the group of consisting of:

a) a pgi gene coding for glucose-6-phosphate isomerase (Pgi, EC No.5.3.1.9), such as, for example, the pgi gene described in U.S. Pat. No.6,586,214 and U.S. Pat. No. 6,465,238 of Corynebacterium glutamicum;

b) an mdh gene coding for malate dehydrogenase (Mdh, EC No. 1.1.1.37),as described for example in WO 02/02778;

c) an mqo gene coding for malate-quinone oxidoreductase (Mqo, EC No.1.1.99.16), as described for example in U.S. Pat. No. 7,094,106 andPCT/EP2005/057216; and

d) an aceE gene (AceE, EC No. 1.2.4.1) coding for the E1p subunit of thepyruvate dehydrogenase complex, as described for example inEP-A-1767616.

These measures may in the case of L-lysine production also be carriedout in addition to the use of lysC^(FBR) alleles and/or overexpressionof one or more genes selected from the group of dapA, dapB, asd, ddh,lysA, aat, lysE and pyc.

The term “attenuation” according to the present invention, describes thereduction or switching off of the intracellular activity of one or moreenzymes (proteins) in a microorganism which are encoded by theappropriate DNA, by for example using a weak promoter or using a gene orallele which codes for a corresponding enzyme with a low activity, orinactivates the corresponding gene or enzyme (protein) and, optionally,combining these measures.

In the case of the AceE polypeptide (see SEQ ID NO: 2 of EP-A-1767616),the attenuation may also be achieved by one or more of the amino acidexchanges selected from the group consisting of a) exchange of Ala atposition 225 for Val, Leu or Ile, preferably Val, b) exchange of Gly atposition 255 for Ser or Thr, preferably Ser, c) exchange of Asn atposition 282 for Gln, and d) exchange of Cys at position 283 for anotheramino acid, preferably Ser, with preference for the following amino acidexchanges selected from the group consisting of e) exchange at position282, f) simultaneous exchange at positions 225 and 283, and g)simultaneous exchange at positions 255 and 283.

The concentration of a protein may be determined by 1- and 2-dimensionalprotein gel fractionation and subsequent optical identification of theprotein concentration in the gel with appropriate analysis software. Acommon method for preparing the protein gels for coryneform bacteria andfor identifying the proteins is the procedure described by Hermann etal. (Electrophoresis, 22:1712-23 (2001)). The protein concentration maylikewise be determined via Western blot hybridization with an antibodyspecific for the protein to be detected (Sambrook et al., Molecularcloning: a laboratory manual. 2^(nd) Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and subsequent optical analysiswith appropriate software for concentration determination (Lohaus andMeyer (1998) Biospektrum 5:32-39; Lottspeich, Angewandte Chemie 38:2476-2492 (1999)). The activity may be determined with the aid of asuitable enzyme assay.

The AmtR regulator, which is also referred to hereinafter as AmtRpolypeptide or AmtR transcription regulator, is a polypeptide having theactivity of a transcription regulator which represses expression of thegenes of nitrogen metabolism when there is a nitrogen excess in thecell. Derepression takes place when there is a nitrogen deficiency.

When a wild-type strain of a coryneform bacterium, preferablyCorynebacterium glutamicum such as, for example, strain ATCC 13032, iscultured in a minimal medium which contains ammonium ions as nitrogensource, there may be a nitrogen deficiency when the ammonium ionconcentration is less than or equal to 5 mM, preferably less than orequal to 1 mM, particularly preferably less than or equal to 0.5 mM.

The degree of adenylation of the signal transduction protein GlnK in thecytoplasm of the coryneform bacterium likewise gives information onwhether there is a nitrogen deficiency or nitrogen excess for the cell.Nitrogen deficiency is present when greater than or equal to 80%,preferably greater than or equal to 90%, particularly preferably greaterthan or equal to 95% of the signal transduction protein is present inadenylated form in the cell.

The genes of nitrogen metabolism which are repressed by the AmtRregulator when there is a nitrogen excess include, inter alia, amtA,amtB, codA, crnT, gdh, glnA, gluA, gltB, ureA and urtA (Table 1 on p.584 in Beckers et al. (Molecular Microbiology 58(2), 580-595 (2005)).

The amino acid sequence of the AmtR regulator of coryneform bacteria isat least 85% or at least 90%, preferably at least 95%, particularlypreferably at least 98% or at least 99% identical to the amino acidsequence of SEQ ID NO:2 and includes or has essentially a length of 222amino acids, with preference for a length of 222 amino acids. An exampleof an AmtR regulator which is at least 98% identical to the amino acidsequence of SEQ ID NO:2 is that of strain ATCC14067. It is listed in SEQID NO:21. It is very particularly preferred for the AmtR regulator toinclude or have the amino acid sequence of SEQ ID NO:2. Whereappropriate, the amino acid sequence of SEQ ID NO:2 or 21 comprises notmore than 3, preferably not more than 2, particularly preferably notmore than one, conservative amino acid exchange(s). The conservativeamino acid exchanges essentially do not alter the activity of the AmtRrepressor.

The term “essentially a length of 222 amino acids” includesconsideration that the length of the encoded polypeptide varies slightlyin different species or strains of L-amino acid-secreting coryneformbacteria through insertion or deletion of one (1) or more, not more than10, 9, 8, 7, 6, 5, 4, 3 or 2, amino acids within the polypeptide or atthe N- or C-terminal end of the polypeptide. One example thereof is theAmtR regulator of Corynebacterium efficiens. The length of thepolypeptide (see SEQ ID NO:11) is in this case 223 amino acids. Thedifferent length of the AmtR regulator is caused by insertion of theamino acid L-glutamic acid between position 151 and 153 of SEQ ID NO:2.

The relation between the amino acids of the amino acid sequence of theAmtR regulator of Corynebacterium efficiens (AmtR Ceff) and the aminoacids of the amino acid sequence of the AmtR regulator ofCorynebacterium glutamicum (AmtR Cglu) is shown below.

AmtR Ceff 1 magavgrprrsaprragknpreeildasaelftrqgfattsthqiadavgAmtR Cglu 1  magavgrprrsaprragknpreeildasaelftrqgfattsthqiadavgAmtR Ceff 51 irqaslyyhfpskteifltllkstvepsmvlagdlanleaspelrlwalvAmtR Cglu 51 irqaslyyhfpskteiflfllkstvepstvlaedlstldagpemrlwaivAmtR Ceff 101 aaevrlllstkwnvgrlyqlpivaseefeeyhtqratltdtfrslateivAmtR Cglu 101 asevrlllstkwnvgrlyqlpivgseefaeyhsqrealtnvfrdlateivAmtR Ceff 151 geddpraelpfhitmsaiemrrndgkvpsplsedslpdtavmladaalavAmtR Cglu 151 g-ddpraelpfhitmsviemrrndgkipsplsadslpetaimladaslav(SEQ ID NO: 11) AmtR Ceff 201 lgadlpgdrvertlellrqadak (SEQ ID NO: 2)AmtR Cglu 200 lgaplpadrvektlelikqadak

It may also be possible to use so-called alignment programs such as, forexample, the ClustalW (Thompson et al., Nucleic Acids Research 25(24),4876-82 (1997)) or MAFFT program (Katoh et al., Nucleic Acid Res.,30:3059-3066 (2002)) for relating the individual amino acids ofdifferent AmtR regulators. The individual amino acids of a polypeptidecan thus be unambiguously related to one another, despite the amino acidsequences formally differing in length.

In the case of aromatic L-amino acids, mutual exchanges ofL-phenylalanine, L-tryptophan and L-tyrosine are referred to asconservative exchanges. In the case of hydrophobic L-amino acids, mutualexchanges of L-leucine, L-isoleucine and L-valine are referred to asconservative exchanges. In the case of polar amino acids, mutualexchanges of L-glutamine and L-asparagine are referred to asconservative exchanges. In the case of basic amino acids, mutualexchanges of L-arginine, L-lysine and L-histidine are referred to asconservative exchanges. In the case of acidic L-amino acids, mutualexchanges of L-aspartic acid and L-glutamic acid are referred to asconservative exchanges. In the case of amino acids containing hydroxylgroups, mutual exchanges of L-serine and L-threonine are referred to asconservative exchanges.

The expression “essentially do not alter the activity of the AmtRrepressor” means that, by the not more than 3 conservative amino acidexchanges, the ability of the AmtR repressor to bind to its DNA bindingsite is altered by not more than 10%, preferably not more than 5%,particularly preferably not more than 1% and very particularlypreferably is unaltered. The activity may be measured by retardation gelelectrophoresis (gel retardation assay) using double-stranded DNAmolecules with the nucleotide sequence of an AmtR binding site of theamtA gene (see SEQ ID NO:14 and 15), of an AmtR binding site of the amtBgene (see SEQ ID NO:16 and 17) or of an AmtR binding site of the gltBgene (see SEQ ID NO:18 and 19), preferably using double-stranded DNAmolecules with the nucleotide sequence of an AmtR binding site of theamtA gene and very particularly preferably using a double-stranded DNAmolecule with the nucleotide sequence of SEQ ID NO:14. The nucleotidesequences are taken from Table 1 of Beckers et al. (MolecularMicrobiology 58(2), 580-595 (2005)).

SEQ ID NO: 1 represents the nucleotide sequence of the coding region ofthe amtR gene of the type strain of Corynebacterium glutamicum(wild-type gene), that is ATCC13032, according to the data in theNational Center for Biotechnology Information (NCBI) database. SEQ IDNO:2 and 4 show the amino acid sequence of the encoded polypeptide. SEQID NO:2 and 4 comprise L-arginine at position 34, L-threonine atposition 87 and L-proline at position 203. It is known that the terminalmethionine can be deleted in the protein synthesis by host-intrinsicenzymes, called amino peptidases. SEQ ID NO:3 additionally indicatesnucleotide sequences located upstream and downstream.

SEQ ID NO: 10 represents the nucleotide sequence of the coding region ofthe amtR gene of Corynebacterium efficiens strain YS-314 according tothe data in the National Center for Biotechnology Information (NCBI)database. SEQ ID NO:11 shows the amino acid sequence of the encodedpolypeptide.

SEQ ID NO: 20 represents the nucleotide sequence of the coding region ofthe amtR gene of Corynebacterium glutamicum ATCC14067. The sequence wasdetermined by the applicant. SEQ ID NO:21 shows the amino acid sequenceof the encoded polypeptide. The amino acid sequence of SEQ ID NO:21comprises L-histidine at position 34, L-isoleucine at position 87 andL-serine at position 203.

The nucleotide sequence of the genome of Corynebacterium glutamicum hasbeen determined by various research groups.

The sequence of the strain ATCC13032 which was determined by Kalinowskiet al. (Journal of Biotechnology 104(1-3), 5-25 (2003)) of BielefeldUniversity (Germany) is available under access number NC_006985. Thename assigned to the amtR gene therein is cg0986 and includes the regionof position 923864-924532 of the complementary strand.

The sequence of the strain ATCC13032 determined by Ikeda and Nakagawa(Applied Microbiology 62(2-3), 99-109 (2003)) of Kitasato University(Japan) is available under the access number NC_003450. The nameassigned to the amtR gene therein is NCgl0828.

The sequence of strain R determined by Yukawa et al. (Microbiology 153(Pt 4), 1042-58 (2007)) of the Research Institute of InnovativeTechnology for the Earth (RITE) (Japan) is available under the accessnumber NC_009342. The name assigned to the amtR gene therein iscgR_0978.

The nucleotide sequence of the genome of Corynebacterium efficiens wasdetermined by Nishio et al. (Genome Research 2003 13(7), 1572-1579(2003)) of Ajinomoto Co. Inc. (Japan). It is available under the accessnumber NC_004369. The name assigned to the amtR gene therein is COG1309Kand includes the region of position 1002436-1003107 of the complementarystrand.

The AmtR regulator belongs to the TetR family of transcriptionregulators and has, like the other members of this family, a typicalhelix-turn-helix motif at the DNA binding site (Ramos et al.,Microbiology and Molecular Biology Reviews 69(2): 326-356 (2003); Jacobyet al., Molecular Microbiology 37(4): 964-977 (2000)).

The nucleotide sequences of the DNA to which the AmtR regulator bindsare known. Jakoby et al. (Molecular Microbiology 37(4), 964-977 (2000))investigated, by deletion analyses, retardation gel electrophoresis (gelretardation assay) and the Matchmaker One-Hybrid system (ClontechLaboratories, Inc., Mountain View, USA), the expression of the amt genewhich codes for the ammonium transporter Amt in Corynebacteriumglutamicum (Siewe et al., Journal of Biological Chemistry 271 (10):5398-5402 (1996)). The ammonium transporter Amt is also referred to as(methyl)ammonium uptake system in Jakoby et al. In Beckers et al.(Molecular Microbiology 58 (2), 580-595 (2005)), the amt gene isreferred to as amtA gene. It has the NCBI access number NCgl1521. Jakobyet al. showed that expression of the amt gene is repressed throughbinding of the AmtR regulator to binding motifs of double-stranded DNAhaving the nucleotide sequence 5′-ATCTATAGAACGATAG-3′ (SEQ ID NO: 22)and 5′-ATCTATAGGCGGATAG-3′ (SEQ ID NO: 23).

Beckers et al. (Molecular Microbiology 58(2), 580-595 (2005)) determinedthe consensus motif of the binding site for the genes regulated by theAmtR regulator. Beckers et al., in contrast to Jakoby et al. (MolecularMicrobiology 37 (4), 964-977 (2000)), indicate the nucleotide sequenceof the reverse complementary DNA strand of the binding site.

Further details on the AmtR regulator may be found inter alia in Walteret al. (Journal of Molecular Microbiology 12, 131-138 (2007)) and A.Burkovski (Archives of Microbiology 179: 83-88 (2003); Article “NitrogenMetabolism and its Regulation” in the “Handbook of Corynebacteriumglutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis,2005).

The term “attenuation” entails reducing the intracellular concentrationor activity of one or more polypeptides (proteins) or enzymes in amicroorganism which are encoded by the appropriate DNA compared with theparent strain. The strain referred to as parent strain or startingstrain is the one on which the attenuation measures have been carriedout. The attenuation can be achieved by reducing the expression of apolypeptide, for example by using a weak promoter, or by using an allelewhich codes for a polypeptide having a lower activity and, whereappropriate, inactivates these measures.

The promoter region of the amtR gene is depicted in SEQ ID NO:5. Thenucleotide sequence of SEQ ID NO:5 is present in SEQ ID NO:3. Position 1of SEQ ID NO:5 corresponds to position 911 of SEQ ID NO:3. Position 90of SEQ ID NO:5 corresponds to position 1000 of SEQ ID NO:3.

In alternative embodiments of the present invention, expression of theAmtR regulator may be reduced by one or more of the modifications of thepromoter region of the amtR gene selected from the group consisting of:

a. replacement of the nucleobase guanine at position 7 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by thymine,

b. replacement of the nucleobase cytosine at position 11 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by guanine,

c. replacement of the nucleobase thymine at position 40 of SEQ ID NO:5by guanine,

d. replacement of the nucleobase thymine at position 45 of the promoterregion of the amtR gene shown in SEQ ID NO:5 by guanine,

e. deletion of one or more of the nucleobases of position 40 to 45,preferably deletion of all nucleobases of position 40 to 45, of thepromoter region of the amtR gene shown in SEQ ID NO:5,

f. deletion of one or more of the nucleobases between position 72 and 78of the promoter region of the amtR gene shown in SEQ ID NO:5, and

g. replacement of one or more of the nucleobases adenine or guaninebetween position 72 and 78 of the promoter region of the amtR gene shownin SEQ ID NO:5 by thymine or cytosine.

Expression of the AmtR regulator may be further reduced according to thepresent invention by exchange of the ATG start codon at position 1 to 3of the coding region of the amtR gene for a GTG or TTG start codon.

In alternative embodiments of the present invention the reduction in theexpression of the amtR gene may diminish the intracellular concentrationof the AmtR regulator to greater than 0% to less than or equal to 75%,greater than 0% to less than or equal to 50%, greater than 0% to lessthan or equal to 25%, greater than 0% to less than or equal to 5%,greater than 0% to less than or equal to 1%, or to greater than or equalto 0.1% to less than or equal to 75%, greater than or equal to 0.1% toless than or equal to 50%, greater than or equal to 0.1% to less than orequal to 25%, greater than or equal to 0.1% to less than or equal to 5%,greater than or equal to 0.1% to less than or equal to 1%, or to greaterthan or equal to 1% to less than or equal to 75%, greater than or equalto 1% to less than or equal to 50%, greater than or equal to 1% to lessthan or equal to 25%, greater than or equal to 1% to less than or equalto 5%, or to greater than or equal to 5% to less than or equal to 75%,greater than or equal to 5% to less than or equal to 50%, greater thanor equal to 5% to less than or equal to 25% of the concentration in theparent strain or starting strain.

In alternative embodiments of the present invention the activity of theAmtR regulator may be reduced by one or more, preferably not more than3, particularly preferably not more than 2, of the amino acid exchangesselected from the group consisting of:

a. exchange of the glycine at position 3 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for another proteinogenic L-amino acid, preferablyL-glutamic acid or L-aspartic acid, particularly preferably L-glutamicacid,b. exchange of the L-isoleucine at position 24 of the amino acidsequence, preferably the amino acid sequence shown in SEQ ID NO:2, SEQID NO:21 or SEQ ID NO:11, for an amino acid selected from the group ofL-asparagine, L-glutamine, L-glutamic acid and L-aspartic acid,preferably L-aspartic acid,c. exchange of the L-leucine at position 31 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-proline,L-asparagine, L-glutamine, L-phenylalanine, L-tyrosine, L-tryptophan,L-glutamic acid and L-aspartic acid, preferably L-proline,d. exchange of the L-phenylalanine at position 32 of the amino acidsequence, preferably the amino acid sequence shown in SEQ ID NO:2, SEQID NO:21 or SEQ ID NO:11, for an amino acid selected from the group ofglycine, L-glutamic acid, L-aspartic acid, L-proline and L-cysteine,preferably L-proline,e. exchange of the glycine at position 36 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-glutamicacid, L-aspartic acid, L-isoleucine, L-histidine and L-phenylalanine,preferably L-histidine, L-glutamic acid or L-aspartic acid, veryparticularly preferably L-aspartic acidf. exchange of the L-threonine at position 42 of the amino acidsequence, preferably the amino acid sequence shown in SEQ ID NO:2, SEQID NO:21 or SEQ ID NO:11, for an amino acid selected from the group ofL-proline, L-isoleucine, L-methionine, L-glutamine, L-tryptophan,L-glutamic acid and L-aspartic acid, preferably L-glutamic acid,g. exchange of the glycine at position 50 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-glutamicacid, L-aspartic acid, L-isoleucine, L-histidine, L-tryptophan andL-phenylalanine, preferably L-tryptophan,h. exchange of the L-glutamine at position 53 of the amino acidsequence, preferably the amino acid sequence shown in SEQ ID NO:2, SEQID NO:21 or SEQ ID NO:11, for an amino acid selected from the group ofL-cysteine, L-methionine, L-tyrosine, L-tryptophan and L-phenylalanine,preferably L-phenylalanine,i. exchange of the L-alanine at position 54 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group ofL-phenylalanine, L-isoleucine, L-tryptophan, L-tyrosine, L-histidine,L-glutamic acid and L-aspartic acid, preferably L-histidine,j. exchange of the L-serine at position 55 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-proline,L-phenylalanine, L-tryptophan, L-lysine, L-arginine, L-glutamic acid andL-aspartic acid, preferably L-phenylalanine,k. exchange of the L-tyrosine at position 57 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-proline,glycine, L-methionine, L-glutamic acid and L-aspartic acid, preferablyL-aspartic acid,l. exchange of the L-tyrosine at position 58 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-proline,L-methionine and L-cysteine, preferably L-proline,m. exchange of the L-histidine at position 59 of the amino acidsequence, preferably the amino acid sequence shown in SEQ ID NO:2 or SEQID NO:11, for an amino acid selected from the group of L-lysine,L-aspartic acid, L-isoleucine, L-proline and glycine, preferablyL-proline, andn. exchange of the L-lysine at position 63 of the amino acid sequence,preferably the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:21 orSEQ ID NO:11, for an amino acid selected from the group of L-alanine,L-glutamic acid, L-aspartic acid, L-asparagine, L-tyrosine andL-tryptophan, preferably L-asparagine.

Preferred embodiments of the present invention may include exchange ofthe glycine at position 3 of the amino acid sequence and exchange of theglycine at position 36 of the amino acid sequence.

In alternative embodiments of the present invention the amino acidexchanges according to the invention may reduce the activity of the AmtRregulator to ranges including greater than 0% to less than or equal to75%, greater than 0% to less than or equal to 50%, greater than 0% toless than or equal to 25%, greater than 0% to less than or equal to 5%,greater than 0% to less than or equal to 1%, or to greater than or equalto 0.1% to less than or equal to 75%, greater than or equal to 0.1% toless than or equal to 50%, greater than or equal to 0.1% to less than orequal to 25%, greater than or equal to 0.1% to less than or equal to 5%,greater than or equal to 0.1% to less than or equal to 1%, or to greaterthan or equal to 1% to less than or equal to 75%, greater than or equalto 1% to less than or equal to 50%, greater than or equal to 1% to lessthan or equal to 25%, greater than or equal to 1% to less than or equalto 5% or to greater than or equal to 5% to less than or equal to 75%,greater than or equal to 5% to less than or equal to 50%, greater thanor equal to 5% to less than or equal to 25% of the activity of the AmtRregulator of the wild type, preferably having the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:11.

In a further embodiment of the present invention it may be additionallypossible to decrease or adjust the expression of the variants accordingto the invention of the AmtR regulator by using known weak promoters.These include inter alia the promoters seqP-RBS_01 to seqP-RBS_07 whichare published in the periodical Research Disclosure under the number512057 (December 2006 edition), and the variants of the dapA promoterdescribed by M. Patek, preferably the variants C7, C13, O1, C2, J2, B31,C5 and B6 (M. Patek in the “Handbook of Corynebacterium glutamicum”(Lothar Eggeling and Michael Bott (editors), CRC Press, Taylor andFrancis Group, Boca Raton, Fla., USA, 2005)).

The attenuation of the amtR gene may be determined with various methods.The concentration can be detected with the aid of one- andtwo-dimensional protein gel fractionation and subsequent determinationof the protein concentration in the gel. A common method for preparingthe protein gels for coryneform bacteria and for identifying theproteins is the procedure described by Hermann et al. (Electrophoresis,22:1712-23 (2001)). The protein concentration can furthermore beanalyzed via Western blot hybridization with an antibody specific forthe protein to be detected (Sambrook et al., Molecular cloning: alaboratory manual. 2^(nd) Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) and subsequent optical analysis withappropriate software for concentration determination (Lohaus and Meyer(1998) Biospektrum 5:32-39; Lottspeich, (1999) Angewandte Chemie 111:2630-2647). The activity of the AmtR regulator as DNA-binding proteincan be measured by retardation gel electrophoresis (Wilson et al. (2001)Journal of Bacteriology 183:2151-2155). This assay is also referred toas the DNA band shift assay. The effect of DNA-binding proteins on theexpression of the genes controlled by them can also be detected byvarious well-described methods of the reporter gene assay (Sambrook etal., Molecular cloning: a laboratory manual. 2^(nd) Ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

In another embodiment, the invention relates to an isolatedpolynucleotide comprising the promoter region or essentially consistingof the promoter region of the amtR gene shown in SEQ ID NO:5 having oneor more of the modifications according to the invention.

In alternative embodiments of the present invention the promoter regionof the amtR gene includes at most 5000, at most 4000, at most 3000, atmost 2000, at most 1000, at most 750, at most 500, at most 250, or atmost 100 nucleobases or base pairs on nucleotide sequences whichnaturally flank the promoter region according to the invention upstreamand downstream.

The term “natural” also includes nucleotide sequences which comprise themutations according to the invention.

In another alternative embodiment the present invention includes anisolated polynucleotide including the coding region or essentiallyconsisting of the coding region of the amtR gene which codes for apolypeptide having the amino acid sequence shown in SEQ ID NO:2 or 21,using a GTG or TTG start codon instead of the ATG start codon.

According to this embodiment, the invention is directed to an isolatedpolynucleotide coding for an AmtR regulator whose amino acid sequencemay be at least 85% or at least 90%, preferably at least 95%,particularly preferably at least 98% or at least 99% and veryparticularly preferably identical to the amino acid sequence of SEQ IDNO:2 and includes or has essentially a length of 222 amino acids,preferably a length of 222 amino acids, and includes one (1) or more,preferably not more than 3, particularly preferably not more than 2, ofthe amino acid exchanges at positions 3, 24, 31, 32, 36, 42, 50, 53, 54,55, 57, 58, 59 and 63 according to the invention.

According to this embodiment, preference may be given to an isolatedpolynucleotide which codes for an AmtR regulator which includes or hasthe amino acid sequence of SEQ ID NO:2 and which includes one (1) ormore, preferably not more than 3, particularly preferably not more than2, of the amino acid exchanges at positions 3, 24, 31, 32, 36, 42, 50,53, 54, 55, 57, 58, 59 and 63 according to the present invention. Inalternative embodiments the amino acid sequence may comprise not morethan 3, preferably not more than 2, particularly preferably not morethan (one) conservative amino acid exchange(s).

A further embodiment of the present invention comprises an isolatedpolynucleotide which codes for an AmtR regulator which includes or hasthe amino acid sequence of SEQ ID NO:11 and which includes one (1) ormore, preferably not more than 3, particularly preferably not more than2, of the amino acid exchanges at positions 3, 24, 31, 32, 36, 42, 50,53, 54, 55, 57, 58, 59 and 63 according to the invention. The amino acidsequence may additionally comprise not more than 3, preferably not morethan 2, particularly preferably not more than (one) conservative aminoacid exchange(s).

In another embodiment of the present invention comprises an isolatedpolynucleotide which codes for an AmtR regulator which includes or hasthe amino acid sequence of SEQ ID NO:21 and which includes one (1) ormore, preferably not more than 3, particularly preferably not more than2, of the amino acid exchanges at positions 3, 24, 31, 32, 36, 42, 50,53, 54, 55, 57, 58, 59 and 63 according to the invention. The amino acidsequence preferably comprises not more than (one) conservative aminoacid exchange(s).

An example of a conservative amino acid exchange is exchange of valineat position 141 of the amino acid sequence for isoleucine.

An additional embodiment of the present invention comprises an isolatedpolynucleotide which codes for an AmtR regulator having the amino acidsequence of SEQ ID NO:2, where the glycine at position 3 is exchangedfor another proteinogenic amino acid, preferably L-glutamic acid orL-aspartic acid, particularly preferably L-glutamic acid. The amino acidsequence of the variant of the AmtR polypeptide which comprisesL-glutamic acid at position 3 is depicted in SEQ ID NO:6 and 8.

Another embodiment of the present invention comprises an isolatedpolynucleotide which codes for an AmtR regulator having the amino acidsequence of SEQ ID NO:2, where the glycine at position 36 is exchangedfor another proteinogenic amino acid, preferably L-histidine, L-glutamicacid or L-aspartic acid, very particularly preferably L-aspartic acid.

An additional embodiment of the present invention comprises an isolatedpolynucleotide which includes or has the nucleotide sequence shown inSEQ ID NO:5 or 7.

Further additional alternative embodiments of the present inventioncomprise an isolated polynucleotide coding for at least a part of theamino acid sequence of the AmtR polypeptide which includes at least 5,at least 10, at least 20, at least 40, at least 80 or at least 100 aminoacids and which comprises at least one amino acid exchange according tothe invention in the AmtR polypeptide, where the mutation leading to theamino acid exchange according to the invention in the polynucleotide isflanked by nucleotide sequences having a length of at most 5000, at most4000, at most 3000, at most 2000, at most 1000, at most 750, at most500, at most 250, or at most 100 nucleobases or base pairs upstream anddownstream, which naturally occurs in coryneform bacteria.

Thus, for example, a polynucleotide having the nucleotide sequence fromposition 500 to 1510 of SEQ ID NO:8 comprises a part of the codingregion of the amtR gene which codes for an amino acid sequence having alength of 170 amino acids, this having the amino acid exchange accordingto the invention at position 3 of the AmtR polypeptide, and a nucleotidesequence having a length of at least 500 nucleobases upstream anddownstream of the mutation leading to the amino acid exchange, as occursnaturally in Corynebacterium glutamicum.

Further embodiments of the present invention comprise vectors whichcomprise the polynucleotides according to the invention.

An embodiment of the present invention comprises cells ofmicroorganisms, especially of bacteria, preferably of the genusCorynebacterium and Escherichia, particularly preferably of the speciesCorynebacterium glutamicum and Escherichia coli, which comprisepolynucleotides or vectors according to the present invention or havebeen produced using polynucleotides or vectors according to the presentinvention.

The polynucleotides according to the invention may be produced by usingclassical in vivo mutagenesis methods with cell populations of bacteriaof the genus Corynebacterium using mutagenic substances such as, forexample, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or ultravioletlight. Subsequently DNA may be prepared or isolated from the mutants,and the corresponding polynucleotide may be synthesized with the aid ofthe polymerase chain reaction (PCR) using primer pairs which allowamplification of the amtR gene or amtR allele, and may be isolated. Itis possible to select for this purpose any primer pairs from thenucleotide sequence located upstream and downstream of the coding regionand of the nucleotide sequence complementary thereto.

Instructions and information on PCR may be found by the skilled personfor example in the handbook “PCR-Strategies” (Innis, Felfand andSninsky, Academic Press, Inc., 1995), in the handbook by Diefenbach andDveksler “PCR Primer—a laboratory manual” (Cold Spring Harbor LaboratoryPress, 1995), in the handbook by Gait “Oligonukleotide synthesis: APractical Approach” (IRL Press, Oxford, UK, 1984) and in Newton andGraham “PCR” (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).Further instructions on PCR may be found for example in WO 06/100177 onpages 15 to 17.

In a further operation, the nucleotide sequence of the polynucleotide isthen determined. This may be determined for example by the chaintermination method of Sanger et al. (Proceedings of the NationalAcademies of Sciences, U.S.A., 74, 5463-5467 (1977)) with themodifications indicated by Zimmermann et al. (Nucleic Acids Research 18,1067 (1990)).

The polypeptide encoded by this nucleotide sequence may then be analyzedfor the amino acid sequence. For this purpose, the nucleotide sequenceis entered in a program for translating DNA sequence into an amino acidsequence. Suitable programs are for example the “Patentin” program whichis obtainable from patent offices, for example the US Patent Office(USPTO), or the “Translate Tool” which is available on the ExPASyProteomics Server in the World Wide Web (Gasteiger et al., Nucleic AcidsResearch 31, 3784-3788 (2003)).

It may also be possible to produce the polynucleotide or amtR alleleaccording to the present invention by in vitro genetic methods.

Suitable methods for in vitro mutagenesis may include inter aliatreatment with hydroxylamine according to Miller (Miller, J. H.: A ShortCourse in Bacterial Genetics. A Laboratory Manual and Handbook forEscherichia coli and Related Bacteria, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1992) or employment of a polymerase chainreaction using a DNA polymerase which shows a high error rate. Such aDNA polymerase is for example the mutazyme DNA polymerase (GeneMorph PCRMutagenesis Kit, No. 600550) supplied by Stratagene (LaJolla, Calif.,USA). A further possibility is to employ mutagenic oligonucleotides asdescribed by T. A. Brown (Gentechnologie für Einsteiger, SpektrumAkademischer Verlag, Heidelberg, 1993) and R. M. Horton (PCR-MediatedRecombination and Mutagenesis, Molecular Biotechnology 3, 93-99 (1995)).The method using the “Quik Change Site-directed Mutagenesis Kit”supplied by Stratagene (La Jolla, Calif., USA), described by Papworth etal. (Strategies 9(3), 3-4 (1996)), may likewise be employed.

The polynucleotides produced by the methods described may be used toproduce recombinant strains of the genus Corynebacterium, preferablyCorynebacterium glutamicum, which comprise the variants according to theinvention of the AmtR regulator and/or comprise the modificationsaccording to the invention in the promoter region of the amtR gene andwhich, compared with the starting or parent strain, release L-aminoacids into the medium surrounding them and/or accumulate them in theinterior of the cell to an increased extent.

A conventional method for incorporating mutations into genes of bacteriaof the genus Corynebacterium, especially of the species Corynebacteriumglutamicum, is that of allele exchange which is also known under thename gene replacement. In this method, a DNA fragment which comprisesthe mutation of interest is transferred into the desired strain, and themutation is incorporated by at least two recombination events orcrossover events into the chromosome of the desired strain, or thesequence of a gene present in the relevant strain is exchanged for themutated sequence.

The DNA fragment comprising the mutation of interest is in this methodtypically present in a vector, in particular a plasmid, which preferablyundergoes only limited, i.e. under selected culture conditions, or noreplication by the strain to be provided with the mutation. In general,a bacterium of the genus Escherichia, preferably of the speciesEscherichia coli, may be used as auxiliary or intermediate host in whichthe vector may be replicated.

Examples of such plasmid vectors are the pK*mob and pK*mobsacB vectorssuch as, for example, pK18mobsacB, which are described by Schafer et al.(Gene 145, 69-73 (1994)), and the vectors described in WO 02/070685 andWO 03/014362. These are replicative in Escherichia coli but not inCorynebacterium. Particularly suitable vectors are those comprising agene with a conditionally negatively dominant effect such as, forexample, the sacB gene (levansucrase gene) of, for example, Bacillus orthe galK gene (galactose kinase gene) of, for example, Escherichia coli.A gene with a conditionally negatively dominant effect is a gene whichunder certain conditions may be disadvantageous, for example toxic, forthe host but, under other conditions, has no negative effects on thehost harboring the gene. A conditionally negative dominant effect genemakes it possible to select for recombinations events in which thevector is eliminated from the chromosome.

In addition, Nakamura et al. (U.S. Pat. No. 6,303,383) have described atemperature-sensitive plasmid for Corynebacterium which is able toreplicate only at temperatures below 31° C. It may likewise be employedfor the purposes of the present invention. The vector may besubsequently transferred into the Corynebacterium by conjugation, forexample by the method of Schäfer (Journal of Bacteriology 172, 1663-1666(1990)) or transformation for example by the method of Dunican andShivnan (Bio/Technology 7, 1067-1070 (1989)). The transfer of the DNAmay also be achieved where appropriate by ballistic methods (e.g.particle bombardment).

Homologous recombination occurring in a first crossover event whichbrings about integration, and of a suitable second crossover event whichbrings about an excision in the target gene or in the target sequenceachieves incorporation of the mutation and results in a recombinantbacterium. The gene in which the desired exchange is to take place isreferred to as target gene.

Methods which may be employed for identifying and characterizing theresulting strains are inter alia those of Southern blottinghybridization, of the polymerase chain reaction, of sequencedetermination, the method of fluorescence resonance energy transfer(FRET) (Lay et al. Clinical Chemistry 43, 2262-2267 (1997)) or methodsof enzymology.

Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991)) used this methodto incorporate a lysA allele which harbored a deletion, and a lysAallele which harbored an insertion, into the chromosome of C. glutamicuminstead of the wild-type gene. Nakagawa et al. (EP 1108790) and Ohnishiet al. (Applied Microbiology and Biotechnology 58(2), 217-223 (2002))employed this method to incorporate various mutations starting from theisolated alleles or polynucleotides into the chromosome of C.glutamicum.

Thus, for example, to incorporate the mutation which leads to exchangeof the amino acid glycine for L-glutamic acid at position 3 of SEQ IDNO:2, preferably as depicted in SEQ ID NO:6 and 8, it may be possible touse a polynucleotide or DNA fragment which includes at least thenucleotide sequence from position 958 to 1058 of SEQ ID NO:8. This DNAfragment comprises the mutation according to the invention and, upstreamand downstream thereof, a nucleotide sequence having a length of in eachcase at least 50 nucleobases.

Preferred DNA fragments according to the present invention have,upstream and downstream of the mutation, a nucleotide sequence having alength of in each case at least about 100, particularly preferably ineach case at least about 250 nucleobases and very particularlypreferably in each case at least about 500 nucleobases. In alternativeembodiments of the present invention the maximum length of thenucleotide sequence located upstream and downstream of the mutation maygenerally be about 500, about 750, about 1000, about 1500, about 2000,about 3000, about 4000 or 5000 nucleobases. Accordingly, in alternativeembodiments of the present invention the total length of thepolynucleotide employed for the allele exchange is not more than about1000, not more than about 1500, not more than about 2000, not more thanabout 3000, not more than about 4000, not more than about 6000, not morethan about 8000 or 10 000 nucleobases.

In the alternative embodiments the output of the bacteria of the genusCorynebacterium and of the fermentation process using the same accordingto the present invention in terms of one or more of the parametersselected from the group of the L-amino acid concentration (L-amino acidproduced per volume), the L-amino acid yield (L-amino acid produced percarbon source consumed), the L-amino acid production (L-amino acidproduced per volume and time) and the specific L-amino acid production(L-amino acid produced per cell dry matter or dry biomass and time orL-amino acid produced per cellular protein and time) or else otherprocess parameters and combinations thereof may be increased by at least0.5%, at least 1%, at least 1.5% or at least 2% based on the startingstrain or parent strain or the fermentation process using the same.

The bacteria produced according to the invention of the genusCorynebacterium may be cultured continuously—as described for example inWO 05/021772—or discontinuously in a batch process (batch cultivation orbatch process) or in a fed batch or repeated fed batch process for thepurpose of producing the desired L-amino acids. A summary of a generalnature about known cultivation methods is available in the textbook byChmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren and periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)). The culture medium or fermentationmedium to be used must satisfy in a suitable manner the demands of therespective strains. Descriptions of culture media for variousmicroorganisms are present in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981). According to the present invention, theterms culture medium and fermentation medium or medium are mutuallyexchangeable.

According to the various embodiments of the present invention, sugarsand carbohydrates such as, for example, glucose, sucrose, lactose,fructose, maltose, molasses, sucrose-containing solutions from sugarbeet or sugar cane production, starch, starch hydrolyzate and cellulose,oils and fats such as, for example, soybean oil, sunflower oil, peanutoil and coconut fat, fatty acids such as, for example, palmitic acid,stearic acid and linoleic acid, alcohols such as, for example, glycerol,methanol and ethanol and organic acids such as, for example, acetic acidor lactic acid may be used as the carbon source.

According to the various embodiments of the present invention, organicnitrogen-containing compounds such as peptone, yeast extract, meatextract, malt extract, corn steep liquor, soybean flour and urea andinorganic compounds such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, may be usedsingly or as mixtures as the nitrogen source.

According to the various embodiments of the present invention,phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used asthe phosphorous source.

The culture medium may additionally comprise salts for example in theform of chlorides or sulfates of metals such as, for example, sodium,potassium, magnesium, calcium and iron, such as, for example, magnesiumsulfate or iron sulfate, which are necessary for growth. Finally,essential growth factors such as amino acids, for example homoserine andvitamins, for example thiamine, biotin or pantothenic acid, may beemployed in addition to the abovementioned substances.

Starting materials may be added to the culture in the form of a singlebatch or be fed in during the cultivation in a suitable manner.

To control the pH of the culture, basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidiccompounds such as phosphoric acid or sulphuric acid may be employed in asuitable manner. The pH may be adjusted to a value of from 6.0 to 8.5,preferably 6.5 to 8. To control foaming, it may be possible to employantifoams such as, for example, fatty acid polyglycol esters. Tomaintain the stability of plasmids, suitable selectively actingsubstances such as, for example, antibiotics may be added to the medium.In order to maintain aerobic conditions, oxygen or oxygen-containing gasmixtures such as, for example, air may be introduced into the culture.In alternative embodiments, liquids enriched with hydrogen peroxide maybe used. The fermentation may be subjected to excess pressure, forexample, an excess pressure of from 0.03 to 0.2 MPa. The temperature ofthe culture may normally be 20° C. to 45° C. and preferably 25° C. to40° C., particularly preferably 30° to 37° C. In batch processes, thecultivation may be continued until a maximum of the desired L-amino acidhas formed, which may normally be achieved within a time range of 10hours to 160 hours. In continuous processes, longer cultivation timesmay be possible. The activity of the bacteria may result in anenrichment (accumulation) of the L-amino acid in the fermentation mediumand/or in the bacterial cells.

Examples of suitable fermentation media may be found inter alia in thepatents U.S. Pat. No. 5,770,409, U.S. Pat. No. 5,840,551 and U.S. Pat.No. 5,990,350 or U.S. Pat. No. 5,275,940.

Analysis of L-amino acids to determine the concentration at one or moretime(s) during the fermentation may be accomplished by separating theL-amino acids by ion exchange chromatography, preferably cation exchangechromatography, with subsequent post-column derivatization usingninhydrin, as described by Spackman et al. (Analytical Chemistry 30:1190-1206 (1958)). It may also be possible to employortho-phtaldialdehyde instead of ninhydrin for the post-columnderivatization. A review article on ion exchange chromatography may befound in Pickering (LC•GC (Magazine of Chromatographic Science) 7(6),484-487 (1989)).

It may likewise be possible to carry out a pre-column derivatization forexample using ortho-phtaldialdehyde or phenyl isothiocyanate, and tofractionate the resulting amino acid derivatives by reversed phasechromatography (RP), preferably in the form of high performance liquidchromatography (HPLC). A method of this type is described for example inLindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)). Detectionmay take place by photometry (absorption, fluorescence).

A summary description of amino acid analysis is to be found inter aliain the textbook “Bioanalytik” by Lottspeich and Zorbas (SpektrumAkademischer Verlag, Heidelberg, Germany 1998).

In various alternative embodiments, the present invention provides aprocess for preparing L-amino acids, preferably L-lysine, L-glutamicacid, L-glutamine, L-arginine, L-proline or L-ornithine, particularlypreferably L-lysine, comprising:

a) fermentation of the cornyeform bacteria according to the invention,preferably of the genus Corynebacterium, particularly preferably of thespecies Corynebacterium glutamicum, in a suitable nutrient medium, and

b) accumulation of the L-amino acid in the nutrient medium and/or in thecells of said bacteria.

The process of the present invention may further comprise the provisionor preparation or isolation of an L-amino acid-containing product inliquid or solid form.

The fermentation process according to the present invention produces afermentation broth which comprises the desired L-amino acid.

A fermentation broth according to the present invention may be afermentation medium or nutrient medium in which a microorganism has beencultivated for a certain time and at a certain temperature. Thefermentation medium or the media employed during the fermentationcomprise(s) all the substances or components which ensure growth of themicroorganism and production of the desired L-amino acid.

When the fermentation according to the present invention is complete,the resulting fermentation broth accordingly comprises

a) the biomass of the microorganism which has been produced as a resultof the growth of the cells of the microorganism,

b) the L-amino acid produced during the fermentation,

c) the organic byproducts produced during the fermentation, and

d) the constituents of the fermentation medium employed or of thestarting materials such as, for example, vitamins such as biotin orsalts such as magnesium sulfate, which have not been consumed by thefermentation.

The organic byproducts may include substances which are produced by themicroorganisms employed in the fermentation in addition to therespective L-amino acid and may include sugars such as, for example,trehalose.

The fermentation broth obtained according to the present invention maybe removed from the culture vessel or fermentation tank, collected, andused to provide an L-amino acid-containing product in liquid or solidform. The expression “obtaining the L-amino acid-containing product” mayalso be used to describe this removal, collection and provision of theL-amino acid-containing product. In a simplest case, the L-aminoacid-containing fermentation broth itself may be the obtained product.

Alternative embodiments of the present invention may include one or moreof the following procedures may be employed to concentrate and/or purifythe L-amino acid product:

a) partial (>0% to <80%) to complete (100%) or virtually complete (≧80%,≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%) removal of the water,

b) partial (>0% to <80%) to complete (100%) or almost complete (≧80%,≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%) removal of the biomass, the latterbeing optionally inactivated before removal,

c) partial (>0% to <80%) to complete (100%) or virtually complete (≧80%,≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, ≧99.3%, ≧99.7%) removal of theorganic byproducts formed during the fermentation, and

d) partial (>0%) to complete (100%) or virtually complete (≧80%, ≧90%,≧95%, ≧96%, ≧97%, ≧98%, ≧99%, ≧99.3%, ≧99.7%) removal of theconstituents of the fermentation medium employed or of the startingmaterials which have not been consumed by the fermentation.

Products having a specified content of L-amino acid may be obtained bythe described procedures.

The partial (>0% to <80%) to complete (100%) or virtually complete (≧80%to <100%) removal of the water (a) may be referred to as drying.

Complete or virtually complete removal of the water, of the biomass, ofthe organic byproducts and of the unconsumed constituents of thefermentation medium obtained according to the present invention providespure (≧80% by weight, ≧90% by weight) or high-purity (≧95% by weight,≧97% by weight, ≧99% by weight) product forms of the L-amino acids.Technical instructions for the procedures a), b), c) or d) areconventionally available and known to skilled artisans.

Four different product forms of the amino acid L-lysine, areconventionally known.

One form of L-lysine-containing products includes concentrated aqueousalkaline solutions of purified L-lysine (EP-B-0534865). A further form,as described for example in U.S. Pat. No. 6,340,486 and U.S. Pat. No.6,465,025, includes aqueous acidic biomass-containing concentrates ofL-lysine-containing fermentation broths. Solid forms include powders orcrystalline forms of purified or pure L-lysine, which may typically bein the form of a salt such as, for example, L-lysine monohydrochloride.A further solid product form is described for example in EP-B-0533039.The product form described therein comprises besides L-lysine most ofthe starting materials used during the fermentative production and notconsumed and, where appropriate, the biomass of the microorganismemployed with a proportion of L-lysine ranging from greater than 0% to100% by weight.

A wide variety of processes appropriate for the various product formsare known to one of ordinary skill in the art for producing theL-lysine-containing product or the purified L-lysine from thefermentation broth.

Methods to produce pure solid L-lysine include ion exchangechromatography, with optional use of activated carbon andcrystallization. The corresponding base or a corresponding salt such as,for example, the monohydrochloride (Lys-HCl) or lysine sulfate(Lys₂-H₂SO₄) may be obtained by this method.

EP-B-0534865 describes a process for producing aqueous basicL-lysine-containing solutions from fermentation broths. In the processdescribed therein, the biomass is separated from the fermentation brothand discarded. A base such as, for example, sodium, potassium orammonium hydroxide is used to adjust the broth to a pH of between 9 to11. The mineral constituents (inorganic salts) are removed from thebroth by crystallization after concentration and cooling, and are eitherused as fertilizer or discarded.

In processes for producing lysine by using the bacteria according to thepresent invention, preferred processes are those resulting in productswhich comprise constituents of the fermentation broth. These may be usedin particular as animal feed additives.

Depending on requirements specified for the product, the biomass may beremoved wholly or partly from the fermentation broth by separationmethods such as, for example, centrifugation, filtration, decantation ora combination thereof, or be left completely therein. Optionally, thebiomass or the biomass-containing fermentation broth may be inactivatedduring a suitable process step, for example by thermal treatment(heating) or by addition of acid.

In alternative embodiments of the present invention, the biomass may becompletely or virtually completely removed so that no (0%) or at most30%, at most 20%, at most 10%, at most 5%, at most 1% or at most 0.1%biomass remains in the prepared product. In further embodiments, thebiomass is not removed, or is removed only in small proportions, so thatall (100%) or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass remainsin the product prepared. In an embodiment according to the presentinvention, accordingly, the biomass may be removed in proportions offrom ≧0% to ≦100%.

The fermentation broth obtained according to the present invention afterthe fermentation, may be adjusted, before or after the complete orpartial removal of the biomass, to an acidic pH with an inorganic acidsuch as, for example, hydrochloric acid, sulfuric acid or phosphoricacid or organic acids such as, for example, propionic acid (GB 1,439,728or EP 1 331 220). It may also be possible to acidify the fermentationbroth with the complete content of biomass. In another embodiment, thebroth may also be stabilized by adding sodium bisulfite (NaHSO₃, GB1,439,728) or another salt, for example an ammonium, alkali metal oralkaline earth metal salt of sulfurous acid.

During the removal of the biomass, organic or inorganic solids presentin the fermentation broth may be optionally partially or completelyremoved. The organic byproducts dissolved in the fermentation broth, andthe dissolved unconsumed constituents of the fermentation medium(starting materials) may remain at least partly (>0%), preferably to theextent of at least 25%, particularly preferably to the extent of atleast 50% and very particularly preferably to the extent of at least 75%in the product. Optionally, the organic byproducts dissolved in thefermentation broth, and the dissolved unconsumed constituents of thefermentation medium may also remain completely (100%) or virtuallycompletely, meaning >95% or >98% or greater than 99%, in the product. Ifa product in this sense comprises at least part of the constituents ofthe fermentation broth, this may also be described by the term “productbased on fermentation broth”.

Subsequently, water may be removed from the broth, or the fermentationbroth thickened or concentrated, by known methods such as, for example,using a rotary evaporator, thin-film evaporator, falling-filmevaporator, by reverse osmosis or by nanofiltration. The concentratedfermentation broth may then be worked up to free-flowing products, inparticular to a fine-particle powder or preferably coarse granules, bymethods of freeze drying, spray drying, spray granulation or by otherprocesses as described for example in the circulating fluidized bedaccording to PCT/EP2004/006655. A desired product may optionally beisolated from the resulting granules by screening or dust removal.

It may also be possible to dry the fermentation broth directly, i.e.without previous concentration by spray drying or spray granulation.

“Free-flowing” is used to describe powders which flow unimpeded out of aseries of glass orifice vessels with orifices of different sizes, atleast out of the vessel with a 5 mm (millimeters) orifice (Klein:Seifen, Öle, Fette, Wachse 94, 12 (1968)). “Fine-particle” describes apowder predominantly (>50%) having a particle size of diameter from 20to 200 μm. “Coarse” describes a product predominantly (>50%) of aparticle size of diameter from 200 to 2000 μm.

The particle size determination may be obtained by methods of laserdiffraction spectrometry. Corresponding methods are described in thetextbook on “Teilchengröβenmessung in der Laborpraxis” by R. H. Müllerand R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996)or in the textbook “Introduction to Particle Technology” by M. Rhodes,published by Wiley & Sons (1998).

The free-flowing, fine-particle powder may be converted by suitablecompaction or granulation processes into a coarse, very free-flowing,storable and substantially dust-free product.

The term “dust-free” describes that the product comprises only smallproportions (<5%) of particle sizes below 100 μm in diameter.

“Storable” in the sense of the present invention describes a productwhich can be stored for at least one (1) year or longer, preferably atleast 1.5 years or longer, particularly preferably two (2) years orlonger, in a dry and cool environment without any substantial loss (atmost 5%) of the respective amino acid occurring.

In a further embodiment, the present invention includes a process, whichis described in principle in DE 102006016158, and in which thefermentation broth obtained using the microorganisms according to thepresent invention, from which the biomass has been optionally removedcompletely or partially, may be further processed according to thefollowing:

the pH is reduced by adding sulfuric acid to 4.0 to 5.2, in particular4.9 to 5.1, and a molar sulfate/L-lysine ratio of from 0.85 to 1.2,preferably 0.9 to 1.0, particularly preferably >0.9 to <0.95, isadjusted in the broth, optionally by adding a further or a plurality ofsulfate-containing compound(s), and

a) the mixture obtained in this way is concentrated by removal of water,and optionally granulated,

where one or both of the following is/are optionally carried out beforea):

c) measurement of the molar sulfate/L-lysine ratio to ascertain therequired amount of sulfate-containing compound(s);

d) addition of a sulfate-containing compound selected from the group ofammonium sulfate, ammonium bisulfate and sulfuric acid in appropriateratios.

Optionally also before b), a salt of sulfurous acid, preferably alkalimetal bisulfite, particularly preferably sodium bisulfite, may be addedin a concentration of from 0.01 to 0.5% by weight, preferably 0.1 to0.3% by weight, particularly preferably 0.1 to 0.2% by weight, based onthe fermentation broth. Preferred sulfate-containing compounds for theabovementioned procedures may include ammonium sulfate and/or ammoniumbisulfate or mixtures of ammonia and sulfuric acid and sulfuric aciditself.

The molar sulfate/L-lysine ratio V may be calculated by the formula:V=2×[SO₄ ²⁻]/[L-lysine].This formula takes account of the fact that the SO₄ ²⁻ anion is doublycharged, or sulfuric acid is dibasic. A ratio of V=1 indicates that thestoichiometric composition Lys₂₋H₂(SO₄) is present, whereas a ratio ofV=0.9 indicates a 10% sulfate deficient composition and a ratio of V=1.1indicates a 10% sulfate excess composition.

It may be advantageous to employ the usual organic or inorganicauxiliaries or carriers such as starch, gelatin, cellulose derivativesor similar substances, as normally used in the processing of foodproducts or feeds as binders, gelling agents or thickeners, or furthersubstances such as, for example, silicas, silicates (EP0743016A) orstearates during the granulation or compaction of the compositionsobtained according to the present invention.

It may be further advantageous to provide the surface of the resultinggranules with oils as described in WO 04/054381. Oils which may be used,include mineral oils, vegetable oils or mixtures of vegetable oils.Examples of such oils are soybean oil, olive oil, soybean oil/lecithinmixtures. In the same way, silicone oils, polyethylene glycols orhydroxyethylcellulose are also suitable. Treatment of the surfaces ofthe granules with oils increases the abrasion resistance of the productgranule and may provide a reduction in the dust content. The oil contentin the product may be from 0.02 to 2.0% by weight, preferably 0.02 to1.0% by weight, and very particularly preferably 0.2 to 1.0% by weight,based on the total amount of the feed additive.

Preferred products have a proportion of ≧97% by weight with a particlesize of from 100 to 1800 μm or a proportion of ≧95% by weight with aparticle size of 300 to 1800 μm diameter. The proportion of dust, i.e.particles with a particle size <100 μm, is preferably less than 1% byweight, particularly preferably not exceeding 0.5% by weight.

Alternatively, the product according to the present invention may beabsorbed on an organic or inorganic carrier conventionally known for theprocessing of feeds, such as, for example, silicas, silicates, meals,brans, flours, starches, sugars or others, and/or be mixed andstabilized with conventional thickeners or binders. Examples of use andprocesses therefore are described in Die Mühle+Mischfuttertechnik, 132(1995) 49, page 817.

In a further alternative embodiment, the product of the presentinvention may be made stable to digestion by animal stomachs, especiallythe stomach of ruminants by coating the granules with film-formers suchas, for example, metal carbonates, silicas, silicates, alginates,stearates, starches, gums and cellulose ethers, as described inDE-C-4100920.

To adjust a desired L-lysine concentration in the product it may bepossible, depending on requirements, to add L-lysine during the processin the form of a concentrate or, where appropriate, of a substantiallypure substance or its salt in liquid or solid form. These can be addedsingly or as mixtures to the resulting or concentrated fermentationbroth, or else during the drying or granulation process.

One embodiment of the present invention further includes a process forproducing a solid L-lysine-containing product, according to descriptionin US 20050220933, and which includes a working up method of thefermentation broth obtained using the microorganisms according to thepresent invention, the working up method comprising:

a) filtration of the fermentation broth, preferably with a membranefilter, to obtain a biomass-containing slurry and a filtrate,

b) concentration of the filtrate, preferably so as to result in a solidscontent of from 48 to 52% by weight,

c) granulation of the concentrate obtained in b), preferably at atemperature of from 50° C. to 62° C., and

d) coating of the granules obtained in c), with one or more of thecoating agent(s). The coating agents preferably used for the coating ind) are selected from the group consisting of

d1) the biomass obtained in a),

d2) a L-lysine-containing compound, preferably selected from the groupof L-lysine hydrochloride or L-lysine sulfate,

d3) a L-lysine-free substance with an L-lysine content of <1% by weight,preferably <0.5% by weight, preferably selected from the group ofstarch, carrageenan, agar, silicas, silicates, meals, brans and flours,and

d4) a water-repellent substance, preferably selected from the group ofoils, polyethylene glycols and liquid paraffins.

The L-lysine content may be adjusted to a specific value by employingone or more of d1) to d4), in particular d1) to d3).

In one preferred embodiment of the present invention the molar ion ratioof the L-lysine-containing products, may be adjusted so that the molarion ratio described by the formula2×[SO₄ ²⁻]+[Cl⁻]−[NH₄ ⁺]−[Na⁺]−[K⁺]−2×[Mg²⁺]−2×[Ca²⁺]/[L-Lys]is from 0.68 to 0.95, preferably 0.68 to 0.90, particularly preferably0.68 to 0.86, as described by Kushiki et al. in US 20030152633.

In the case of L-lysine according to the present invention, the solidproduct according to alternative embodiments comprises a lysine content(as lysine base) of from 10% by weight to 70% by weight or 20% by weightto 70% by weight, preferably 30% by weight to 70% by weight and veryparticularly preferably from 40% by weight to 70% by weight, based onthe dry matter of the product. Maximum lysine base contents may be 71%by weight, 72% by weight, or 73% by weight. The water content of theL-lysine-containing solid product may be up to 5% by weight, preferablyup to 4% by weight, and particularly preferably less than 3% by weight.

The invention claimed is:
 1. An isolated polynucleotide comprising thepromoter region of the amtR gene shown in SEQ ID NO:5 and wherein theamtR gene is attenuated by at least one of a) replacement of the guanineat position 7 of SEQ ID NO:5 by thymine, b) replacement of cytosine atposition 11 of SEQ ID NO:5 by guanine, c) replacement of thymine atposition 40 of SEQ ID NO:5 by guanine, d) replacement of thymine atposition 45 of SEQ ID NO:5 by guanine, e) deletion of one or more of thenucleobases of position 40 to 45 of SEQ ID NO:5, f) deletion of one ormore of the nucleobases between position 72 and 78 of SEQ ID NO:5, andg) replacement of one or more of the nucleobases adenine or guaninebetween position 72 and 78 of SEQ ID NO:5 by thymine or cytosine.
 2. Avector comprising a polynucleotide as claimed in claim
 1. 3. An isolatedmicroorganism cell comprising the vector as claimed in claim
 2. 4. Anisolated microorganism cell comprising the polynucleotide as claimed inclaim
 1. 5. The isolated polynucleotide of claim 1, wherein the amtRgene is attenuated by replacement of the guanine at position 7 of SEQ IDNO:5 by thymine.
 6. The isolated polynucleotide of claim 1, wherein theamtR gene is attenuated by replacement of cytosine at position 11 of SEQID NO:5 by guanine.
 7. The isolated polynucleotide of claim 1, whereinthe amtR gene is attenuated by replacement of thymine at position 40 ofSEQ ID NO:5 by guanine.
 8. The isolated polynucleotide of claim 1,wherein the amtR gene is attenuated by replacement of thymine atposition 45 of SEQ ID NO:5 by guanine.
 9. The isolated polynucleotide ofclaim 1, wherein the amtR gene is attenuated by deletion of one or moreof the nucleobases of position 40 to 45 of SEQ ID NO:5.
 10. The isolatedpolynucleotide of claim 1, wherein the amtR gene is attenuated bydeletion of all nucleobases of position 40 to 45 of SEQ ID NO:5.
 11. Theisolated polynucleotide of claim 1, wherein the amtR gene is attenuatedby deletion of one or more of the nucleobases between position 72 and 78of SEQ ID NO:5.
 12. The isolated polynucleotide of claim 1, wherein theamtR gene is attenuated by replacement of one or more of the nucleobasesadenine or guanine between position 72 and 78 of SEQ ID NO:5 by thymineor cytosine.